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Considerations of Solid-Phase DNA Amplification Ramkumar Palanisamy,† Ashley R. Connolly,† and Matt Trau* Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane Qld-4072, Australia. Received November 10, 2009; Revised Manuscript Received February 3, 2010
Solid-phase (SP) polymerase chain reaction (PCR) is an increasingly popular tool used to produce immobilized DNA for a variety of applications, including high-throughput DNA sequencing and SNP analysis. Despite its usefulness, the mechanism of DNA amplification using immobilized primers has not been thoroughly explored. Herein, we describe a SP-PCR process that was designed to explore and better understand some limitations of SP-DNA amplification. The rate of SP-DNA amplification was measured, and the ability to exponentially amplify DNA on a surface was demonstrated. Approximately 50 amol of DNA was amplified to detectable levels using SP-PCR. The mechanism and some limitations of the reaction were investigated by measuring the density of the primer on the surface prior to amplification and the amount of immobilized amplicon produced after SP-PCR. This enabled some of the practical limitations of the reaction to be addressed within a logical theoretical framework.
INTRODUCTION
Table 1. PCR Primer and DNA Template Sequences
The polymerase chain reaction (PCR) has revolutionized the field of molecular biology by enabling a specific fragment of DNA in a heterogeneous mixture to be amplified in excess of one million fold. This very sensitive technique enables DNA to be amplified to levels that are detectable and useful in a wide range of biological applications (1, 2). Conventionally, PCR is performed in solution using two primers that are each designed to hybridize to the 3′ ends of a denatured DNA duplex. A thermostable DNA polymerase is used to extend the 3′ end of each primer to replicate the duplex, which accumulates exponentially with repetitive rounds of thermal cycling. PCR can also be used to generate an immobilized amplicon in a process called solid-phase (SP) PCR. There are two common forms of SP-PCR. In one form, DNA amplification is initiated with both primers in solution to enable the amplicon to be produced in an amount sufficient to extend an immobilized primer (3-6). The second form involves generating an immobilized amplicon by grafting both primers on a surface (6, 7). The primer(s) can be immobilized on a microtiter plate (8, 9), a flat surface (10-12), or a microbead encased in a micelle. In each case, enzymatic extension of the primer produces a tethered amplicon that can be easily detected, quantified, and manipulated for subsequent applications including cloning, high-throughput DNA sequencing and SNP detection (13, 14). Despite its potential, the amount of immobilized amplicon produced using SP-PCR is limited. Although the surface itself reduces the yield of amplicon, numerous nonfouling surfaces and strategies to project the primer away from the surface have had only limited success (15, 16). To better understand the factors that reduce the yield of amplified DNA, a SP-PCR assay on silica microbeads was developed. This enabled the mechanism of SP-DNA amplification to be thoroughly investigated. The detection limit of the SP-PCR system was determined, and the kinetics of DNA amplification on the surface of a microbead * Correspondence to Matt Trau,
[email protected], Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane Qld-4072. Phone: +61 7 3346 4173, fax: +61 7 3346 3973. † These authors contributed equally to this work.
Oligonucleotide Sequences (5′-3′) Forward primer Primer probe DNA Template
Reverse primer
NH2 TTG ATC CTG GGA GTT GAC AGC CAA AGA TGG GTC CAG TTC A Cy5-T GAA CTG GAC CCA TCT TTG GCT GTC AAC TCC CAG GAT CAA GGC ATC CAC GGG CGC ATC TCC GTT GAG GGC CAG GAA GTG AAC TGG ACC CAT CTT TGG ALEXA 488-GAA ACC CAT CAG TCA AGG AGG CAT CCA CGG GCG CAT CTC C
were investigated. The amount of immobilized primer extended during the amplification reaction was also quantified and the accuracy of the assay was assessed. These data provide an insight into the mechanism and some of the fundamental limitations of SP-DNA amplification.
MATERIALS AND METHODS Fluorescently Encoded Microbeads. Preparation of 6 µm diameter silica microbeads has been previously described (17). The microbeads were coated with aminopropylsilane, and the surface was derivatized with adipic acid (18). Coupling of Oligonucleotides to Microbeads. The 5′ end of the forward primer was coupled to 6 µm adipic acid coated microbeads using standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry (18). The DNA coupled microbeads were pelleted by centrifugation at 10 000 G for 30 s and washed three times with 1 mL of 0.1 M 2-morpholinoethane sulfonic acid (MES buffer supplemented with 0.01% SDS) and stored at 4 °C. Oligonucleotides. Unlabeled oligonucleotides were purchased from GeneWorks (GeneWorks Pty. Ltd., Adelaide, Australia), and fluorescently labeled oligonucleotides were purchased from Invitrogen (Invitrogen Corporation, California, USA). The oligonucleotides used in this study are listed in Table 1. Solid-Phase Polymerase Chain Reaction. 50 pg of singlestranded (ss) DNA template was amplified in a 30 µL reaction containing 1.25 U of Taq DNA polymerase (GoTaq Flexi DNA polymerase, Promega Corporation, Wisconsin, USA), 0.2 mM
10.1021/bc900491s 2010 American Chemical Society Published on Web 03/23/2010
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Figure 1. SP-PCR. Microbeads containing the immobilized forward primer were suspended in a solution containing the reverse primer and the DNA template. The template annealed to the immobilized forward primer (1) enabling the 3′ end of the primer to be extended by Taq polymerase (2) to generate dsDNA. The DNA duplex was thermally denatured (3) to allow the reverse primer to anneal to the newly synthesized immobilized amplicon. (4). Extension of the reverse primer regenerated dsDNA duplex (5) which was subjected to repetitive cycles of denaturation, annealing, and extension to produce many immobilized amplicons. The extent of SP-PCR was determined by denaturing the amplicon and probing the surface immobilized ssDNA with a fluorescent reverse primer. The level of fluorescence was quantified using a flow cytometer.
of each dNTPs, 2.5 mM MgCl2, 1× PCR buffer (GoTaq PCR buffer, Promega), 0.1% Tween 20, 0.5 µM of A488 reverse primer, and approximately 4 × 104 microbeads containing the immobilized forward primer. Cycling was carried out in a Corbett Research thermocycler using the following conditions: denaturation at 94 °C for 1 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 2 min, and 72 °C for 1 min. DNA Hybridization. Following PCR, the immobilized amplicon was denatured at 100 °C for 90 s in 400 µL of 4× SSC buffer (0.6 M NaCl, 0.06 M sodium citrate). The microbeads were pelleted and denaturation was repeated twice. 50 ng of A488 reverse primer was hybridized to the immobilized ssDNA in 10 µL milli Q and 4 µL hybridization buffer (50 mM NaHPO3, 0.5 M NaCl, and 0.5% SDS). Hybridization was performed at 55 °C for 60 min in a vessel shaking at 1400 rpm. The microbeads were then washed and suspended in 100 µL of 0.01% SDS in preparation for analysis using flow cytometry. Flow Cytometry. Fluorescence on the surface of the microbeads was measured using a DakoCytomation Moflo flow cytometer. The Alexa 488 (A488) fluorophore (Invitrogen Corporation, CA, USA) was excited with a 488 nm laser and emission monitored at 530 nm. The Cy5 fluorophore (GE Healthcare Bio-Sciences, Uppsala, Sweden) was excited with a 635 nm laser and emission monitored at 670 nm. Data were collected on a logarithmic scale and analyzed using Summit v 4.0 software.
RESULTS Assay Design. The mechanism of SP-PCR was investigated using a PCR assay designed to exclusively amplify surface immobilized DNA. The assay was initiated by hybridization of a ssDNA template to an immobilized forward primer. Enzymatic extension of the immobilized primer generated an immobilized amplicon. A reverse primer could then hybridize to the immobilized amplicon and undergo extension to produce a copy of the ssDNA template (Figure 1). Therefore, SP-PCR will only progress after enzymatic extension of the immobilized forward primer. This enabled the mechanism of SP-PCR to be analyzed in the absence of any amplification of DNA in solution, which will inevitably occur if amplification is initiated with a dsDNA template.
Following amplification, the amount of PCR product on the surface was measured by denaturing the amplicon and hybridizing a fluorescently labeled DNA probe to the immobilized ssDNA. The intensity of the fluorescent signal on surface of the microbead was measured using a flow cytometer to reveal the amount of SP-DNA synthesis. Assay Optimisation. Several parameters likely to affect SPPCR were optimized to ensure efficient DNA amplification. Washing the microbeads with PCR buffer prior to amplification was essential for successful SP-PCR. The concentration of magnesium was varied between 2 and 3 mM and amplification was optimal at 2.5 mM. To ensure amplification was not limited by the amount of the reverse primer in the reaction, its concentration was the doubled from 0.5 µM which produced only 15% increase in the amount of immobilized amplicon. Subsequent increases in concentration had no effect on the yield of SP-DNA amplification. 4 × 104 microbeads were used in the SP-PCR. The amount of forward primer in the PCR was changed by varying the number of microbeads added to the reaction. Halving the number had no significant effect on the yield of the immobilized amplicon, while an excess number of microbeads reduced the yield of immobilized amplicon, which was attributed to sedimentation of the microbeads. The rate of hybridization of a complementary strand of DNA to the immobilized primer will dictate the duration of the annealing step during PCR. This was determined by directly hybridizing a complementary fluorescent probe to the immobilized forward primer at 55 °C in PCR buffer. The extent of hybridization to the immobilized primer was determined by measuring the fluorescence using flow cytometry. After hybridization for 1.5 min, the fluorescence on the surface of the microbead reached 70% of the maximum level measured after 10 min. This indicated an annealing time of approximately 2 min during SP-PCR cycling was sufficient for the DNA template to hybridize to the immobilized primer. Detection Limit. Fluorescently encoded microbeads have been used for sensitive detection of PCR products (19-22). However, amplification of DNA directly on a surface is considerably less sensitive (6, 7). Therefore, we investigated some factors that were likely to influence SP-DNA amplification
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template (To) amplifies with an efficiency (Et) over (j) cycles to produce an amount of product (Tj) according to eq 1 (23). Tj ) To(1 + Et)j
Figure 2. Replicate SP-PCR were prepared each containing approximately 4 × 104 microbeads with the forward primer immobilized on their surface, the reverse primer in solution, and the specified amount of DNA template. Following amplification, the immobilized dsDNA was denatured and the extent of SP-PCR was determined by hybridizing a fluorescent probe to the immobilized ssDNA amplicon. Values represent the mean fluorescence ( standard deviation of triplicate reactions (y-axis) for each template concentration (x axis) which was determined by flow cytometric analysis of the microbeads.
in an attempt to characterize and better understand the limitations of the reaction. The detection limit and analytical range of this SP-PCR was assessed by amplifying serial dilutions of the DNA template (from 100 ng to 100 fg) in triplicate reactions containing the immobilized forward primer and the reverse primer in solution. The extent of SP-DNA amplification was determined by hybridizing a fluorescent probe to the ssDNA immobilized on the surface after denaturation of the amplicon. The fluorescence increased with template concentration (Figure 2), indicating that more DNA amplification occurred on the surface at higher template concentration. When the DNA template was present in excess of 1 ng, there was no significant increase in the amount of immobilized amplicon produced, suggesting the surface became saturated with the with PCR products. The coefficient of variation of the replicate reactions varied from 4% to 20% and was, on average, 10% throughout the analytical range of template concentrations. Amplification Mode. The amount of SP-PCR product increased as more DNA template was added to the reaction (Figure 2). However, it is unclear if this increase was the result of linear amplification or inefficient exponential amplification of the template. When a single primer is used in a PCR, the amount of template will only double after each cycle resulting in linear amplification of DNA. In the presence of both primers, DNA amplification progresses exponentially and the amount of amplicon produced is maximized. Therefore, the mode of SP-DNA amplification was investigated by omitting the reverse primer to ensure linear amplification of the template. The reverse primer was then added to a replicate reaction to enable it to proceed exponentially. The extent of DNA amplification was determined by hybridizing a fluorescent probe to the ssDNA remaining on the surface after denaturation of the amplicon. After 25 cycles of linear amplification, there was a 2.3-fold increase in the amount of PCR product on the surface. After 25 cycles of exponential PCR, there was a 7.8-fold increase in the amount of PCR product, which was 3.4-fold more than linear amplification (Figure 3). This demonstrates that the reverse primer is utilized during SP-PCR to increase the amount of SPDNA produced during amplification. The change in fluorescence on the surface of the bead at different cycle numbers was used to determine the efficiency of SP-DNA amplification. In a PCR, the initial amount of DNA
(1)
Equation 1 was solved for different values of Et to produce a curve that approximated the experimentally acquired data (Figure 3). This revealed that exponential SP-PCR progressed with an amplification efficiency (Et) of approximately 9%, before the reaction reached a plateau after 17 cycles. Quantification. The amplification efficiency of SP-PCR is approximately 91% lower than PCR performed in solution. This inefficiency may be attributed to a low primer concentration, its inability to diffuse through solution, or inefficient enzymatic extension. However, attempts to overcome these inefficiencies by projecting primers away from the surface or modifying surface have had only limited success (15, 16). In an attempt to better understand the factors that limit SP-PCR, the amount of DNA amplification on the surface was accurately measured. Initially, the amount of forward primer immobilized on the surface of each microbead was quantified. Different amounts of a fluorescently labeled DNA strand complementary to the immobilized primer were prepared and incubated with replicate samples containing 4 × 104 microbeads. The fluorescence on the surface of each bead was plotted as a function of DNA concentration to produce a curve indicative of a Langmuir isotherm (24) (Figure 4). The amount of immobilized primer was determined by calculating the amount of fluorescent DNA that saturated the surface of 4 × 104 microbeads. A 6.0 ( 0.5 µm diameter microbead was found to contain (2.6 ( 0.2) × 106 primers or (2.3 ( 0.3) × 1010 primers per mm2. Therefore, approximately 0.17 pmol of forward primer was added to each PCR with the addition of 4 × 104 microbeads. The amount of immobilized amplicon produced after 30 cycles of SP-PCR was also measured. Following amplification, the immobilized amplicon was denatured to generate immobilized ssDNA. Different amounts of the fluorescently labeled
Figure 3. Replicate SP-PCRs were prepared each containing approximately 4 × 104 microbeads with the forward primer immobilized on their surface and 1 ng of DNA template. The reverse primer was either omitted from the SP-PCR (dashed line) or added to the reaction (solid line). Amplification progressed for the indicated number of cycles. Following amplification, the immobilized dsDNA was denatured, and the extent of SP-PCR was determined by hybridizing a fluorescent probe to the immobilized ssDNA amplicon. Values represent the mean fluorescence ( standard deviation of triplicate reactions (y-axis) for each template concentration (x axis), which was determined by flow cytometric analysis of the microbeads. The efficiency of exponential SP-PCR was calculated by solving eq 1 for different values of Et. An amplification efficiency of 9% (Et ) 0.09) and an arbitrary To value of 72.17 were substituted into eq 1 to calculate Tj at different cycle numbers to obtain a line (dotted) that best fits the experimental data.
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bead can ideally contain (3.5 ( 0.4) × 1010 amplicons per mm2. Therefore, only (24 ( 4)% of the available surface was covered with the amplicon after 30 cycles of SP-PCR.
DISCUSSION
Figure 4. Replicate samples containing 4 × 104 microbeads with the forward primer immobilized on the surface were prepared. Each sample was hybridized with the specified concentration of fluorescently labeled oligonucleotide probe (]), complementary to the immobilized primer. The experiment was repeated (0) and performed with fewer microbeads (2 × 104 (∆)). The fluorescence on the surface of each bead was measured using flow cytometry. The values represent the average fluorescence on the surface of each microbead (y-axis) for each concentration of the complementary fluorescent probe (x-axis) measured by flow cytometry. The amount of immobilized primer was determined by calculating the amount of fluorescent DNA that saturated the surface of the microbeads.
Figure 5. SP-PCR amplification of 1 µg of DNA template was performed in replicate reactions containing 2 × 104 microbeads, then the immobilized amplicons were denatured. Each sample was hybridized with the specified concentration of fluorescently labeled oligonucleotide probe (]), complementary to the immobilized amplicon. The experiment was repeated (∆) and performed with fewer microbeads (1 × 104 (0)). The fluorescence on the surface of each microbead was measured using flow cytometry. The values represent the average fluorescence on the surface of each microbead (y-axis) for each concentration of the complementary fluorescent probe (x-axis). The amount of immobilized amplicon produced by SP-PCR was determined by calculating the amount of fluorescent DNA that saturated the surface of the microbeads.
reverse primer were prepared and incubated with replicate samples containing 2 × 104 microbeads. The fluorescence on the surface of each microbead after hybridization is depicted in Figure 5. The amount of immobilized PCR product was measured and it was calculated that each microbead contained (9.5 ( 0.7) × 105 amplicons, or (8.4 ( 1.1) × 109 amplicons per mm2. Therefore, only (36 ( 7)% of the primers immobilized on the surface were extended after 30 cycles of SP-PCR. The molecular geometry of ssDNA in solution has been modeled. The theoretical radius of gyration of a 96 base ssDNA amplicon was calculated to be approximately 3.0 nm with a cross section area of 28.8 nm2. Therefore, each 6.0 ( 0.5 µm
We have devised a SP-PCR assay on a microbead that enabled the mechanism of SP-DNA amplification to be explored. The assay described herein was performed using only the reverse primer in solution to generate an immobilized amplicon. This enabled the mechanism of SP-PCR to be analyzed in the absence of any amplification of DNA in solution. DNA was amplified exponentially on the surface and the amount of amplicon produced was quantified. Reaction conditions for optimal SP-DNA amplification were elucidated and found to be similar to those of conventional PCR, despite the reported use of lengthy cycling parameters (5). Conditioning the DNA-coated surface with the PCR reaction buffer was essential for successful amplification and hybridization of the template to the immobilized primer occurred rapidly during thermal cycling. The forward primer was found to be securely immobilized on the surface despite reports of primer detachment during thermal cycling (6). The stability was attributed to the covalent attachment of the forward primer and the molecular stability of the silica microbeads. As a result, the coefficient of variation was on average 10% between replicate amplification reactions. This level of reproducibility was obtained without agitating the microbeads during thermal cycling, which has been reported to retard SP-DNA amplification (25). It is unclear if the microbeads remain in suspension during the reaction, but it is clear that they all contain a consistent amount of surface immobilized amplicon. Therefore, the template was not locally sequestered on the surface but diffused freely in solution initiating DNA polymerization on different microbeads in the reaction. The amount of PCR product generated on the surface of each microbead increased when more template was added to the reaction (Figure 2). However, when the template concentration was in excess of 1 ng there was no appreciable increase in the amount of amplicon produced. This may be due to the DNA template saturating the surface or other factors may be acting to retard amplification. It is generally accepted that lack of diffusion, proximity to the surface, and low concentration of the immobilized primer most likely inhibit SP-DNA amplification (15, 16, 25, 26). However, Taq polymerase was enzymatically active while in close proximity to the surface and was able to perform both forward and reverse priming reactions effectively. Moreover, the DNA template was shown to undergo exponential amplification during the reaction. The efficiency of exponential SP-PCR was approximately 9% (i.e., Et ) 0.09) indicating that only 9 of 100 primers were extended during each cycle of PCR. This is substantially lower than solution PCR where the efficiency approaches 90-100% (i.e., Et ) 0.9-1.0) (27). Attempts to increase the overall yield of the immobilized amplicon by increasing PCR cycle number and reducing the number of microbeads had no measurable impact on the detection limit. Approximately 3 × 107 copies of DNA could be amplified to detectable levels by SP-PCR. Only a fraction (36%) of the immobilized primers were extended during SP-PCR. This suggests amplification was not limited by the amount of the immobilized primer, but more likely by the generation of the amplicon itself. The immobilized amplicon (96 bases) was 2.4-fold longer than the primer (40 bases) and could conceivably “mask” adjacent primers after amplification. More specifically, primers within approximately one gyration radius of the amplicon would experience this masking effect. This will predominate when the primers are immobilized at high density, while at low density, amplification will be limited by insufficient primers.
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ACKNOWLEDGMENT We gratefully acknowledge funding and support from the NBCF through the National Collaborative Breast Cancer Research Grant Program.
LITERATURE CITED
Figure 6. Neighboring DNA interactions during SP-PCR. (A) When the DNA amplicons (black line) are at low density, the 3′ end of the immobilized primer (arrow) will undergo extension by Taq polymerase to produce a new immobilized amplicon. (B) As SP-PCR progresses, the amplicon density increases and it is proposed that extension of the immobilized primer is retarded because the DNA template (gray line) will hybridize to an adjacent amplicon in preference to the primer.
A 96-base amplicon has a theoretical gyration radius of 3.0 nm and a cross-sectional area of approximately 28.8 nm2. This would enable approximately 3.5 × 1010 amplicons to be produced per mm2, on the surface of each 6 µm bead. However, only 8.4 × 109 amplicons were generated per mm2, which is substantially lower than expected. The amount of product produced during SP-PCR may not be limited exclusively by primer “masking”, other factors may also retard amplification. SP PCR progressed exponentially, but amplification was retarded during the later cycles (Figure 3) which suggests the progress of the reaction was inhibiting amplification. As the amount of immobilized PCR product increased, the DNA template may be likely to hybridize to the immobilized amplicon in preference to the primer. This will prevent primer extension and limit the amount of SP-DNA amplification. This inhibitory effect is more likely to occur when the immobilized primer and amplicon are in close proximity. More specifically, when separated by the approximate gyration diameter of the template, the PCR template is more likely to hybridize to the immobilized amplicon in preference to the immobilized primer (Figure 6). Under these conditions, it is predicted that SP-DNA amplification will reach a “plateau” when the immobilized amplicon density is 75% lower than that predicted theoretically. This is equivalent to (8.7 ( 1.0) × 109 amplicons per mm2, which is similar (97 ( 17%) to the experimentally determined value of (8.4 ( 1.1) × 109 amplicons per mm2. The inhibitory effect of SP-PCR is likely to be enhanced by factors that increase the gyration radius of the amplicon. Larger amplicons have an increased gyration radius and are predicted to have a lower yield during SP-PCR, which is consistent with experimental findings (28). “Spacers” designed to project the primers from the surface will make the primers more accessible to the polymerase, but will also increase the gyration radius of the amplicon and may limit the yield of SP-PCR. In summary, we have characterized some factors that influence SP-DNA amplification and have identified other factors that are likely to limit the yield of DNA during amplification. It has been demonstrated that DNA polymerase retained catalytic activity in close proximity to a surface. SP-PCR was used to exponentially amplify ssDNA to generate a unique homogeneous amplicon on a surface. However, the amplification efficiency was substantially lower than that of solution-phase PCR. The amount of amplicon produced was not exclusively limited by a lack of diffusion and concentration of the immobilized primer, but more likely by the production of the immobilized amplicon during the reaction. The data presented here provide insight into the mechanism of SP-DNA amplification and highlight some factors that may need to be addressed in order to achieve efficient DNA amplification on a surface.
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